B-Lactam Antibiotics - Penicillins PDF

Al-Zaytoonah University of Jordan Faculty of Pharmacy MedChem 3 Course #: 0201511 3 Credit Hours β-Lactam Antibiotics - Penicillins 2023-2024 Agenda Penicillins What we need to know? ① General Structure, pharmacophoric features ② Mechanism of action ③ Synthesis ④ SAR ⑤ Main Problems ⑥ How to imp...

Al-Zaytoonah University of Jordan Faculty of Pharmacy MedChem 3 Course #: 0201511 3 Credit Hours β-Lactam Antibiotics - Penicillins 2023-2024 Agenda Penicillins What we need to know? ① General Structure, pharmacophoric features ② Mechanism of action ③ Synthesis ④ SAR ⑤ Main Problems ⑥ How to improve drug design to tackle problems: in vitro stability, administration route (oral is better), stability towards β –lactamases, spectrum of activity (antibacterial activity) 2023-2024 Introduction: β – Lactam Antibiotics Antibiotics that possess the β – Lactam ring (a four-membered cyclic amide) such as penicillins and cephalosporins. This group of antibiotics have: 1) Broad spectrum of antibacterial action 2) Potent and rapid bactericidal action against bacteria in the growth phase. 3) Low frequency of toxic and other side effects in host. 2023-2024 Ring Types and Numberings of Clinically Available Βeta-lactam Antibiotics Memorize these! 2023-2024 The First β-Lactam Antibiotics: Penicillins  Penicillins are the most widely used class of antibiotics.  Penicillins inhibit bacterial cell wall synthesis  Discovered by Fleming from a fungal colony (1928) shown to be nontoxic and antibacterial. First used on humans in 1942 (US, Streptococcal septicemia).  The original fermentation-derived penicillins were produced by growth of the fungus Penicillium chrysogenum. 2023-2024 Penicillin G Fermentation-derived Active against G+ mainly General Structure and Biosynthesis of Penicillin Two prototypes: 4 5 7 1 3 2 Memorize this structure for 6-APA in the blue box. Benzyl penicillin: (2S,5R,6R)-3,3-Dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid. 2023-2024 Semisynthetic Penicillins Synthesis of 6-APA from penicillin G 6-aminopenicillanic acid (6-APA) is synthesized by hydrolyzing naturally produced Penicillin G. Then 6-APA is used to synthesize other penicillins (semisynthetic because it relied on a naturally-occurring precursor). 2023-2024 Then 6-APA is used to synthesize other “improved” penicillins Peptidoglycan Structure of Bacterial Cell Walls is the Target for Penicillins  β – Lactams bind at the active site of the transpeptidase enzyme that cross-links the peptidoglycan strands at the positions indicated by arrows on the figure above.  It does this by mimicking the D-alanyl-D-alanine residues that would normally bind to this site. 2023-2024 Mechanism of Action of Penicillin  Cross-linking of bacterial cell walls inhibited by penicillin The enzyme responsible for the crosslinking reaction is known as the transpeptidase enzyme .  β – Lactams including penicillin irreversibly inhibit the enzyme transpeptidase by reacting with a serine residue in the transpeptidase  inhibit cell wall synthesis.  Cell walls are essential for bacteria to live. 2023-2024 Penicillin Mimics the Natural Substrate Antimetabolite for acyl-d-Ala-d-Ala Figure. Comparison of penicillin, 6-substituted penicillins, and acyl-d-Ala-d-Ala. 2023-2024 Mechanism of Transpeptidase Cross-linking and Inhibition by Penicillins 2023-2024 β-Lactam Ring Acting as an Acylating Agent 2023-2024 Why β-Lactam Carbonyl Groups Are Required for Antibiotics Activity? Compare with Tertiary Amide 2023-2024 The 3D Shape of Penicillin  Penicillin contains a highly unstable bicyclic system consisting of a four-membered β –lactam ring fused to a five-membered thiazolidine ring.  The skeleton of the molecule suggests that it is derived from amino acids cysteine and valine.  The overall shape of the molecule is like a half-open book, as shown on the left. 2023-2024 Penicillin Is Also a Substrate for β-Lactamases A problem that may lead to bacterial resistance 2023-2024 Major SAR Considerations, Chemical and Biological Instabilities 1) Hydrolysis by beta-lactamases (penicllinases in case of penicillins) 2) Instability in basic media or in presence of nucleophiles (e.g., OH-) 3) Instability in acids 2023-2024 1) Deactivation by Beta-lactamases Mechanism of β-lactamase Deactivation of Penicillin: 2023-2024 You need to know this! 2) Instability of Βeta-lactams to Nucleophiles Hydrolysis in basic media 2023-2024 3) Instability of Penicillins in Acid Hydrolysis in Acidic media Hydrolysis involves the C-6 side chain. Skip Mechanism just remember how it is related to the the C-6 side chain. 2023-2024 Ring Opening of Penicillins Leads to Loss of Antibiotic Activity 2023-2024 SAR Rules Structural Modifications to Solve the Main Problems of Penicillins: 1) 2) 3) 4) Deactivation by beta-lactamases Oral Bioavailability Spectrum of activity PK properties/Prodrugs 2023-2024 Reduce Sensitivity to Beta-lactamases Fig. β-Lactamase resistant/sensitive structural features. 2023-2024 Making Penicillins β-Lactamase Resistant 2023-2024 2) Making Penicillins Orally Active (i.e., resistant to acidic hydrolysis) 2023-2024 How Improve Acid Stability to Allow Oral Administration 1) Pen V 2) Amino Penicillins 2023-2024 Orally Active, Acid and β-Lactamase Resistant 2023-2024 Modify Spectrum of Activity - BROAD SPECTRUM PENICILLINS (1) Aminopenicillins (2) Carboxypenicillins 2023-2024 (3) Ureidopenicillins Broad-spectrum penicillins  Ureidopenicillins (Figure above) are the newest class of broad-spectrum penicillins and have a urea functional group at the α-position.  Generally, they have better properties than the carboxypenicillins and have largely replaced them in the clinic. 2023-2024 Broader Spectrum of Activity - INCREASED ACTIVITY AGAINST G- BACTERIA Add hydrophilic groups on the side chains of R on the 6th position of the lactam ring  Hydrophilic groups on the 6th position side chain have little effect on G+ bacteria (e.g., penicillin T), or cause a reduction of activity (e.g., penicillin N).  However, they lead to an increased activity against Gbacteria. 2023-2024 Prodrugs to Aid Absorption of Ampicillin Through the Gut Wall Mechanism of hydrolysis of prodrugs: 2023-2024 Prodrugs: Polar Extended Ester for Penicillin G 2023-2024 Synergism of Penicillins with Beta Lactamse Inhibitors Clavulanic Acid 2023-2024 Synergism of Penicillins with Probencid Probencid  Probenecid is a moderately lipophilic carboxylic acid that can block facilitated transport of penicillin through the kidney tubules and slows down the rate at which penicillin is excreted.  Probenecid also competes with penicillin for binding sites on albumin. As a result, penicillin levels in the bloodstream are enhanced and the antibacterial activity increases—a useful tactic if faced with a particularly resistant bacterium. 2023-2024 Examples on Commercially Significant Penicillins 2023-2024 Examples on Commercially Significant Penicillins 2023-2024 Key Points: Penicillins ① Penicillins have a bicyclic structure consisting of a β-lactam ring fused to a thiazolidine ring. ② The strained β-lactam ring reacts irreversibly with the transpeptidase enzyme responsible for the final cross-linking of the bacterial cell wall. ③ Penicillin analogues can be prepared by fermentation or by a semi-synthetic synthesis from 6-aminopenicillanic acid. ④ Variation of the penicillin structure is limited to the acylside chain or -OMe on 6th position carbon of the beta lactam ring. ⑤ Penicillins can be made more resistant to acid conditions by incorporating an electronwithdrawing group into the acyl side chain. ⑥ Steric shields can be added to penicillins to protect them from bacterial β-lactamase enzymes. ⑦ Broad spectrum activity is associated with the presence of an α-hydrophilic group on the acyl side chain of penicillin. ⑧ Prodrugs of penicillins are useful in masking polar groups and improving absorption from the gastrointestinal tract. ⑨ Extended esters are used which undergo enzyme-catalysed hydrolysis to produce a product which degrades spontaneously to release the penicillin. ⑩ Probenecid can be administered with penicillins to hinder the excretion of penicillins. 2023-2024 Al-Zaytoonah University of Jordan Faculty of Pharmacy MedChem 3 Course #: 0201511 3 Credit Hours β-Lactam Antibiotics - Cephalosporins 2023-2024 Agenda Cephalosporins What we need to know? ① Pharmacophore structure ② General Structures (highlighted in lecture) ③ SAR ④ How to improve: in vitro stability, administration route (oral is better), stability towards β –lactamases, spectrum of activity (antibacterial activity) ⑤ Important Mechanisms of action, or reaction mechanisms. 2023-2024 Cephalosporin C 5 7 4 6 8 1 3 2 Amino Adipic Acid IUPAC NAME: (6R,7R)-3-[(Acetyloxy)methyl]-7-{[(5R)-5-amino-5carboxypentanoyl]amino}-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 2023-2024 Mechanism of Inhibition of Transpeptidase by Cephalosporins 2023-2024 Biosynthesis of Cephalosporins Building Blocks/Precursors 2023-2024 Positions for Possible Modifications of Cephalosporin C 5 4 6 8 1 3 2 The shading indicates positions which can be varied 2023-2024 First Generation Cephalosporins Cephalothin Acetyloxy group at position 3 is readily hydrolyzed by esterase enzymes to give the less active alcohol. thiophene Metabolic Hydrolysis of Cephalothin: 2023-2024 First Generation Cephalosporins Cephaloridine Cefalexin Orally absorbed  Cephaloridine exists as a zwitterion and is soluble in water, but, like most first generation cephalosporins, it is poorly absorbed through the gut wall and has to be injected. Pyridinium at C3 in Cephaloridine is metabolically stable. The pyridine can still act as a good leaving group for the inhibition mechanism, but is not cleaved by esterases.  The Me at C3 in Cefalexin increases absorption through the gut and is stable to metabolism  orally bioavailable. 2023-2024 Other First Generation Cephalosporins Modifications on two positions: R and X Oral Parenteral Cefazolin Cephalexin Cefadroxil Second Generation Cephalosporins: Cephamycins Cephamycin C Cefoxitin Urethrane group Urethrane group  Cephamycins contain a methoxy substituent at position 7, which has proved advantageous. Modification of the side chain gave cefoxitin, which showed a broader spectrum of activity than most first-generation cephalosporins. This is due to greater resistance to β–lactamase enzymes, which may be due to the steric hindrance provided by the methoxy group.  Cefoxitin showed good metabolic stability to esterases owing to the presence of the urethane group at position 3, rather than an ester. 2023-2024 Second Generation: Oximinocephalosporins Urethrane group  Oximinocephalosporins has been a major advance in cephalosporin research. These structures contain an iminomethoxy group at the α –position of the acyl side chain, which significantly increases the stability of cephalosporins against the β -lactamases produced by some organisms (e.g. Haemophilus influenza ).  Increased resistance to β -lactamases and mammalian esterases (like cefoxitin). Unlike cefoxitin, cefuroxime retains activity against streptococci and, to a lesser extent, against staphylococci. 2023-2024 Examples: Second Generation Modifications on 4 positions: R, X, Y and Z Parenteral Cefuroxime Cefoxitin Oral Cefotetan Cefaclor Cefprozil 2023-2024 Third Generation Cephalosporins Aminothiazole ring in Ceftazidime enhances the penetration of cephalosporins through the outer membrane of Gram-negative bacteria, and may also increase affinity for the transpeptidase enzyme. As a result, third-generation cephalosporins containing this ring have a marked increase in activity against these bacteria. 2023-2024 Third Generation Cephalosporins Iminomethoxy Aminothiazole (R)  Other 3rd generation compounds: cefotaxime, ceftizoxime, and ceftriaxone, with different substituents at position 3 to vary the pharmacokinetic properties. They play a major role in antimicrobial therapy because of their activity against Gram-negative bacteria, many of which are resistant to other β –lactams.  As such infections are uncommon outside hospitals, physicians are discouraged from prescribing these drugs routinely and they are viewed as ‘reserve troops’ to be used for troublesome infections which do not respond to the more commonly prescribed β lactams. 2023-2024 Examples: Third Generation Cephalosporins 2023-2024 Third Generation Cephalosporins 2023-2024 Fourth Generation Cephalosporins Iminomethoxy Aminothiazole  They are zwitterionic compounds having a positively charged substituent at position 3 and a negatively charged carboxylate group at position 4. This property appears to radically enhance the ability of these compounds to penetrate the outer membrane of Gram negative bacteria.  They are also found to have a good affinity for the transpeptidase enzyme and a low affinity for a variety of β-lactamases. 2023-2024 Prodrug Cephalosporins NOCH3 O HN H H O S N O OCONH2 O O CH3 O CH3 Cefuroxime axetil O NOCH3 HN N H2N S H H O S N O O O Cefpodoxime proxetil 2023-2024 OCH3 O CH3 O O CH3 CH3 Another Prodrug: “Fifth-generation” Cephalosporin The 1,3-thiazole ring is thought to be important for its activity against MRSA. Ceftaroline fosamil Newest Cephalosporin “Parenteral Drugs” Has activity against various strains of MRSA and multi-resistant Streptococcus pneumonia (MDRSP). 2023-2024 Resistance to Cephalosporins  The activity of a specific cephalosporin against a particular bacterial cell is dependent on the same factors as those for penicillins. i.e. the ability to reach the transpeptidase enzyme, stability to any β -lactamases which might be present, and the affinity of the antibiotic for the target.  For example, most cephalosporins (with the exception of cephaloridine) are stable to the β -lactamase produced by S. aureus and can reach the transpeptidase enzyme without difficulty. Therefore, the relative ability of cephalosporins to inhibit S. aureus comes down to their affinity for the target transpeptidase enzyme. Agents such as the cephamycins and ceftazidime have poor affinity, whereas other cephalosporins have a higher affinity.  The MRSA organism contains a modified transpeptidase enzyme ( PBP2a) for which both penicillins and cephalosporins have poor affinity. 2023-2024 Key Points: Cephalosporins  Cephalosporins contain a strained β-lactam ring fused to a dihydrothiazine ring.  In general, first-generation cephalosporins offer advantages over penicillins in that they have greater stability to acid conditions and β-lactamases, and have a good ratio of activity against Gram-positive and Gram-negative bacteria. However, they have poor oral availability and are generally lower in activity. Mainly reserved for parenteral formulations.  Variation of the 7-acylamino side chain alters antimicrobial activity, whereas variation of the side chain at position 3 predominantly alters the metabolic and pharmacokinetic properties of the compound. Introduction of a methoxy substitution at C-7 is possible, making compounds more resistant to beta lactamases.  Semisynthetic cephalosporins can be prepared from 7-aminocephalosporanic acid (7-ACA). 7-ACA is obtained from the chemical hydrolysis of natural cephalosporins. 2023-2024 Key Points: Cephalosporins  Deacetylation of acetyl groups on 3rd position of cephalosporins occurs metabolically to produce inactive metabolites. Metabolism can be blocked by replacing the susceptible acetoxy group with metabolically stable groups.  A methyl substituent at position 3 is good for oral absorption but bad for activity unless a hydrophilic group is present at the α-position of the acyl side chain.  Cephamycins are cephalosporins containing a methoxy group at position 7.  Oximinocephalosporins have resulted in several generations of cephalosporins with increased potency and a broader spectrum of activity, particularly against Gram-negative bacteria. 2023-2024 Al-Zaytoonah University of Jordan Faculty of Pharmacy MedChem 3 Course #: 0201511 3 Credit Hours Other Beta Lactam Antibiotics 2023-2024 Carbapenems We will use IUPAC Numbering:  In general, carbapenems have the broadest spectrum of activity of all the β-lactam antibiotics and inactivate β-lactamases. It combines in one molecule the functional features of the best of the β-lactam antibiotics as well as the β-lactamase inhibitors.  The sulfur atom is now attached to C-3 as part of a functionalized side chain. The endocyclic olefi nic linkage also enhances the reactivity of the β-lactam ring. Both make thienamycin unstable, and this caused difficulties in the original isolation studies. 2023-2024 Carbapenem Drugs and SAR Rules Natural & unstable Not clinical analog Not orally active Clinical analog Inhibitor of Dehydropeptidase-1  Carbapenems are don’t have the sulphur atom and 6-acylamino found in the bicyclo ring system of penicillins and cephalosporins. The stereochemistry of the side chain at position 6 is also opposite—another factor contributing to β-lactamase resistance.  Thienamycin the first of the carbapenems, was isolated from Streptomyces cattleya.  Imipenem is semisynthetic and clinical analog. Chemically more stable than thienamycin but susceptible to metabolism by renal dehydropeptidase-1 which hydrolyze the βlactam and deactivates it. Serious side effects include seizures especially with large dose.  Useful combination: An inhibitor for this enzyme, cilastatin, is coadministered with imipenem to protect it. The drug penetrates most tissues well except the CSF. 2023-2024 Carbapenems with 4-β-Methyl  The 4-β-methyl group confers stability toward dehydropeptidase-1. All used as single agents for treating susceptible infections. Orally available and injections.  Meropenem is resistant to metabolism by a dehydropeptidase enzyme because of the different substituent at position 2.  Ertapenem has better stability against dehydropeptidase-1 while the ionized benzoic acid contributes to high protein binding and prolongs the half-life of the drug allowing once-daily dosing. Not active against Pseudomonas.  Doripenem is the newest member. Active against Pseudomonas. 2023-2024 Carbapenem Prodrugs oral prodrug carbapenem  Tebipenem is the first carbapenem whose prodrug form, the pivalyl ester, is orally available  Tebipenem is formulated as the ester tebipenem pivoxil which helps in better absorption and improved bioavailability.  It was developed as a replacement drug to combat resistant bacteria.  It has performed well in clinical trials for ear infection and looks likely to be further developed in future. Currently it is only marketed in Japan. 2023-2024 Monobactams  Nocardicins are were isolated from natural sources. None of the natural monobactams is clinically useful but inspired the synthesis of Aztreonam.  Aztreonam is a totally synthetic parenteral antibiotic whose antimicrobial spectrum is devoted almost exclusively to gram-negative microorganisms, and it can inactivate some β-lactamases  the α-oriented methyl group at C-2 is associated with the stability of aztreonam toward β-lactamases. The sulfamic acid moiety attached to the β-lactam ring was unprecedented. However, the principal side chain on 3rd position closely resembles that of ceftazidime.  Nearly unchanged by metabolism. Aztreonam is given by injection and is primarily excreted in the urine. The drug is well tolerated, and adverse effects are infrequent. Cross-allergenicity with penicillins and cephalosporins has not often been reported  The primary clinical use of aztreonam is against severe infections caused by gram-negative microorganisms, especially those acquired in the hospital. 2023-2024 Al-Zaytoonah University of Jordan Faculty of Pharmacy MedChem 3 Course #: 0201511 3 Credit Hours  Beta-lactamase Inhibitors 2023-2024 1) Clavulanic Acid  Weak and unimportant antibiotic activity  Powerful and irreversible inhibitor of most β -lactamases, which means that it is used as a sentry drug. in combination with traditional penicillins, such as amoxicillin. 2023-2024 Clavulanic Acid Acting as a Suicide Inhibitor for Most Β-Lactamases 2023-2024 Clavulanic Acid Acting as a Suicide Inhibitor for Most Β-Lactamases – Cont.  Clavulanic acid also fits the active site of β –lactamase, and the β -lactam ring is opened by the serine residue in the same manner. However, the acyl-enzyme intermediate then reacts further with another enzymatic nucleophilic group (possibly NH 2) to bind the drug irreversibly to the enzyme.  The mechanism requires the loss or gain of protons at various stages, and an amino acid, such as histidine, in the active site would be capable of acting as a proton donor/acceptor. Drugs that operate in this way are often called mechanism- based inhibitors or suicide substrates because the enzyme is committing suicide by reacting with them. The great advantage of this approach is that the alkylating agent is generated at the site where it is meant to act and is, therefore, highly selective for the target enzyme. If the alkylating group had not been disguised in this way, the drug would have alkylated the first nucleophilic group it met in the body and would have shown little, or no, selectivity. 2023-2024 2) Penicillanic Acid Sulphone Derivatives In combination : Ampicillin/sulbactam “Unasyn” Parenteral “Sultamicillin” Oral Sulbactam pivoxil is a prodrug of sulbactam. Sulbactam is poorly adsorbed from GI tract. Sulbactam pivoxil has a better absorption than the parent drug and provides high serum levels after oral administration. In combination “Zosyn”: Piperacillin/tazobactam I.V. First-line therapy for the treatment of bloodstream infections in neutropenic cancer patients. Sultamicillin  Act as suicide substrates for β -lactamase enzymes and have similar properties.  Sulbactam has a broader spectrum of activity against β -lactamases than clavulanic acid 2023-2024 but is less potent. 3) Olivanic Acids (example on carbapenems) MM 13902  The olivanic acids (e.g. MM 13902 ) were isolated from strains of Streptomyces olivaceus and are carbapenem structures like thienamycin.  They are very strong inhibitors of β -lactamase—in some cases 1000 times more potent than clavulanic acid.  They are also effective against the β -lactamases which break down cephalosporins and are unaffected by clavulanic acid.  Due to lack chemical stability and poor penetration into the bacterial cell they were not further pursued for development. 2023-2024